The gut microbiota may contribute to colorectal cancer etiology by modulating luminal metabolism of organic and xenobiotic compounds and by inducing immunologic and structural changes in the gut epithelium. In particular, trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite of dietary choline and L- carnitine, obtained from red meat and other animal foods, has been associated with an elevated risk of colorectal cancer, as well as cardiovascular disease and diabetes. The only prospective study on circulating TMAO and incident colorectal cancer reported a significant 2- to 3-fold increased risk for women with high TMAO, high choline or low betaine status. These findings warrant urgent replication in other prospective data, including in men and in various racial/ethnic groups. The TMAO association with various metabolic conditions also calls for studies to elucidate the likely downstream mechanisms, as well as to characterize the associated gut bacterial profiles in populations with diverse genetic makeup and dietary exposures. We propose to assess prospectively the association of plasma choline metabolites with colorectal cancer risk in the Multiethnic Cohort (MEC) Study, utilizing specimens archived in 2001-2006 from over 67,000 adults of five racial/ethnic groups. Also, we propose to investigate the association of plasma TMAO with mechanism-based biomarkers and with gut microbial profiles in a subset of 2,000 MEC participants who are being characterized in detail for their exposome biomarkers, gut microbiome, genome, and body fat distribution as part of an ongoing Program Project (P01) on obesity. Specifically (Specific Aim 1), we propose to conduct a nested case-control analysis of pre-diagnostic plasma TMAO, choline, betaine, and carnitine for the 1,160 incident MEC colorectal cancer cases, accrued between 2001 (age 53-83y) and 2015, and matched controls. We also propose (Specific Aim 2) to evaluate the likely involvement of inflammation, insulin resistance and growth factors, dyslipidemia, and oxidative stress by analyzing fasting plasma TMAO in 2,000 healthy MEC participants (~200 per sex-ethnic group) on whom biomarkers for the above mechanisms will have been analyzed as part of the ongoing MEC- P01. For Specific Aim 3, we will explore the gut microbial community profiles that are associated with plasma TMAO and identify the related functional composition of the metagenome using a computational approach. We will make efficient use of the detailed gut microbiota phylogeny data being generated in the 2,000 MEC-P01 participants. We hypothesize that high TMAO levels are associated with low bacterial diversity and an altered composition and distribution of bacterial phylotypes. We will account for the potential modifying effects of diet (red meat intake, plasma choline status) and host genetics (FMO3), again utilizing the food frequency questionnaire responses and genome-wide SNP data available on the P01 participants. This study will cost- effectively evaluate the importance of gut microbial metabolite related to red meat consumption, an established risk factor for colorectal neoplasms, and may contribute to improved risk profiling and new intervention strategies.